Protein 4.1 is a cytoskeletal protein critical for the normal shape and strength of the red cell membrane. It binds to spectrin and actin to stabilize the spectrin skeleton, and attaches to the cytoplasmic domains of integral membrane proteins. Red cell protein 4.1 (p4.1R) is a specialized member of a heterogeneous family of isoforms that arise from a single gene by highly regulated pathways of tissue specific alternative pre-mRNA splicing. Two major splicing events alter expression and function of p4.1R in erythroid progenitors: induction of the inclusion of exon 16, which encodes a critical peptide within the spectrin-actin binding domain; and suppression of the inclusion of exon 2' which removes an upstream translation start site, causing erythroid cells to produce the 80kd form, but not the 135 kd forms found in many other tissues. Exon 16 inclusion is prominent in muscle and testes, but not in most other tissues. During the past five years, we have localized five critical RNA target sequences (cis elements): within exon 16, in an atypical 5' splice site, in the first 25 bases in the upstream intron and more distantly in both the upstream and downstream intron. The intronic sequences behave as enhancers, and the exonic elements as silencers. We have also developed cell-free splicing systems from HeLa cells and mouse erythroleukemia cells (MELC) that mimic the erythroid behavior of added pre-mRNA, and we have also identified a 40 kd protein by UV crosslinking that may be a trans-acting splicing factor involved in exon 16 regulation. We now propose to characterize each element in detail for their precise contribution, and to identify, isolate, and functionally characterize the transacting splicing factors that interact with them. We shall isolate RNA binding proteins, using the target sequences as probes, and then the proteins that bind to them, using the yeast two hybrid system. The expression during, and impact on, erythropoiesis of these proteins will then be assessed. A similar sequence of studies will be performed to delineate elements controlling exon 2/2' selection after further characterization of the 5' end of the p4.1R gene, which we have found to be far more complex than previously believed. In this manner, we hope to elucidate the regulation of protein 4.1 splicing and the physiological role that factors governing tissue-specific pre-mRNA splicing play in erythropoiesis.